Inhibition of Salmonella enterica Serovar Typhimurium Motility and Entry into Epithelial Cells by a Protective Antilipopolysaccharide Monoclonal Immunoglobulin A Antibody

Abstract

Secretory immunoglobulin A (SIgA) antibodies directed against the O antigen of lipopolysaccharide (LPS)are the primary determinants of mucosal immunity to gram-negative enteric pathogens. However, the underlying mechanisms by which these antibodies interfere with bacterial colonization and invasion of intestinal epithelial cells are not well understood. In this study, we report that Sal4, a protective, anti-O5-specific monoclonal IgA, is a potent inhibitor of Salmonella enterica serovar Typhimurium flagellum-based motility. Using video light microscopy, we observed that Sal4 completely and virtually instantaneously "paralyzed" laboratory and clinical strains of serovar Typhimurium. Sal4-mediated motility arrest preceded and occurred independently of agglutination. Polyclonal anti-LPS IgG antibodies and F(ab) 2 fragments were as potent as was Sal4 at impeding bacterial motility, whereas monovalent Fab fragments were 5-to 10-fold less effective. To determine whether motility arrest can fully account for Sal4's protective capacity in vitro, we performed epithelial cell infection assays in which the requirement for flagellar motility in adherence and invasion was bypassed by centrifugation. Under these conditions, Sal4-treated serovar Typhimurium cells remained noninvasive, revealing that the monoclonal IgA, in addition to interfering with motility, has an effect on bacterial uptake into epithelial cells. Sal4 did not, however, inhibit bacterial uptake into mouse macrophages, indicating that the antibody interferes specifically with Salmonella pathogenicity island 1 (SPI-1)-dependent, but not SPI-1-independent, entry into host cells. These results reveal a previously unrecognized capacity of SIgA to "disarm" microbial pathogens on mucosal surfaces and prevent colonization and invasion of the intestinal epithelium.

Rapid Effects of a Protective O-Polysaccharide-Specific Monoclonal IgA on Vibrio cholerae Agglutination, Motility, and Surface Morphology

“…There is emerging evidence to suggest that protective anti-LPS IgA antibodies may have direct effects on microbial virulence, independent of agglutination (20)(21)(22)(23)(24). In the case of V. cholerae, Bishop and Camilli demonstrated using dark-field microscopy that polyclonal anti-LPS IgA antibodies inhibit V. cholerae motility in vitro, an observation that has been confirmed by others (13,14).…”

“…The monoclonal dimeric IgA antibodies Sal4 (specific for the Salmonella enterica serovar Typhimurium O5 antigen) (29) and 2D6 (specific for V. cholerae OPS) (12) were obtained from Marian Neutra (Children's Hospital, Boston, MA). Sal4 and 2D6 hybridomas were maintained in CD hybridoma serum-free, protein-free medium (Gibco-Invitrogen, Carlsbad, CA) without antibiotics at 37°C in a 5% CO 2 -95% air atmosphere, as described previously (23). The concentrations of Sal4 and 2D6 were determined by sandwich enzyme-linked immunosorbent assay with IgA TEPC-15 (Sigma-Aldrich, St. Louis, MO) as a standard.…”

“…Visually, IgA-mediated motility arrest of V. cholerae occurred before the bacteria became agglutinated, suggesting that the two phenomena are distinct. In the case of Salmonella enterica serovar Typhimurium, we reported in 2008 that treatment of S. Typhimurium with a protective O-antigen-specific IgA monoclonal antibody (MAb) known as Sal4 results in the immediate arrest of flagellum-based motility, independent of agglutination (23). More recently we demonstrated that Sal4 triggers a reduction in type 3 secretion system (T3SS) activity and alterations in outer membrane integrity and onset of exopolysaccharide (EPS) production.…”

O-Antigen-NegativeSalmonella entericaSerovar Typhimurium Is Attenuated in Intestinal Colonization but Elicits Colitis in Streptomycin-Treated Mice

“…Additionally, the ⌬wbaP mutant (SKI12) showed a severe defect in motility. There are several other reports connecting LPS mutations with a loss in motility: in serovar Typhimurium, addition of a monoclonal anti-LPS antibody to a diluted bacterial culture led to instant paralysis of the bacteria (8), and in a Pseudomonas aeruginosa ⌬waaL strain, a severe impairment in motility was detected (2). Also, an Escherichia coli mutant lacking O antigen was reported to show a nonmotile phenotype (37).…”

RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE

“…Swimming assays were performed on 0.3% agar plates as described in ref. 60 with slight modifications; see SI Appendix, Materials and Methods for details. Swarming assays were performed on 0.5% agar plates.…”